The major long-term objective of our research is to understand the detailed molecular mechanisms by which proteins recognize and interact with specific DNA sequences. We are approaching this problem through a detailed structural and functional study of type II restriction and modification systems. The EcoRI system is the best characterized. X-ray crystallographic analysis of the endonuclease is in progress, and we have determined the gene and amino acid sequences of both enzymes. We plan to explore complementary contacts between the enzymes and their substrate sequence. The importance of specific amino acid contacts will be examined by using site directed mutagenesis to alter the nucleotide sequence and characterizing the resulting altered enzymes. Contacts on the DNA surface will be identified through substrate protection experiments. Enzyme-DNA complexes will be treated with chemical reagents, such as dimethyl sulfate or ethyl nitrosourea, or with DNase to determine the pattern of protection from cleavage when the endonuclease or methylase is bound to the substrate sequence. We plan to continue to facilitate the X-ray crystallographic analysis by further improvement of strains which produce the enzymes and by assisting in large scale enzyme purification. Two other type II systems will be examined in order to determine the generality of information derived from the EcoRI system. The EcoRII endonuclease and methylase have been partially characterized. The substrate sequence provides a variation from the perfect two-fold rotational symmetry of the EcoRI system. An isoschizomer of EcoRI has been identified in Rhodopseudomonas sphaeroides: another protein recognizing the same substrate sequence as the EcoRI enzymes should provide a particularly interesting comparison. Recognition of defined sequences in DNA is an essential process in DNA replication, recombination and the expression of the genome during cellular metabolism and development. Elucidation of mechanisms involved in sequence-specific interactions utilizing relatively simple systems should illuminate these complex processes.